The present invention is concerned with a method for obtaining amniotic mesenchymal tissue cells (AMTC) and/or chorionic mesenchymal tissue cells (CMTC) as well as UCC, with cells obtainable by these methods and the use of the cells and preparations.
Cells derived from the amniotic membrane of human placenta have been receiving particular attention because of their stem cell potentiality and immunomodulatory properties, which make them an attractive candidate source for cell therapy approaches.
In this study, we have isolated cells from the mesenchymal layer of amnion (AMTC) and studied their suppressant and stimulator characteristics. We found that unfractionated AMTC can inhibit T cell allogeneic proliferation responses even in the absence of direct cell contact, which points to the existence of suppressor soluble factor(s). In addition, we have identified two populations of AMTC discordant for expression of the HLA-DR, CD45, CD14, and CD86 cellular markers. While unfractionated, HLA-DR+ and HLA-DR− AMTC populations fail to induce proliferation of resting allogeneic T cells. HLA-DR+ AMTC induced strong proliferation of anti-CD3 primed allogeneic T cells in cell-cell contact settings. The revelation that cell populations from human amnion mesenchyme possess both suppressive and stimulatory properties sheds additional light on the immunomodulatory functions of this tissue and its critical role in fetal-maternal tolerance and supports the quest for cells with similar characteristics among mesenchymal cells from other tissues, including mesenchymal stromal cells (MSC).
The amnion and chorion mesenchymal layers from human term placenta harbor cells that present with fibroblast-like morphology, have clonogenic potential, display multi-potent differentiation capacity including osteogenic, adipogenic, chondrogenic, vascular, endothelial, cardiomyocyte, skeletal muscle lineages. [Bailo, 2004 #31; Zhao, 2005 #97; Portmann-Lanz, 2006 #96; Parolini, 2006 #64; Alviano, 2007 #93]. These characteristics are reminiscent of the properties described for bone marrow derived mesenchymal stromal cells (BM-MSC), a much more extensively characterized cell type which is gaining increasing interest for clinical applications [Dominici, 2006 #109; Le Blanc, 2006 #50; Rasmusson, 2006 #91].
Beside bone marrow, where they have been first described [Friedenstein, 1968 #51], other sources for MSC have been reported such as adipose tissue [Zuk, 2001 #65; Kern, 2006 #69; Lee, 2004 #107], cord blood [Romanov, 2003 #66; Lee, 2004 #68] peripheral blood [Villaron, 2004 #67; Roufosse, 2004 #74], amniotic fluid [De Coppi, 2007 #87].
One critical characteristic of MSC is their ability to suppress T cell proliferation in MLR [Di Nicola, 2002 #11; Krampera, 2003 #12; Le Blanc, 2003 #22; Ueta, 2002 #17; Barry, 2005 #88] in addition to other immunomodulatory properties, such as their induction of Th2 responses, up regulation of T regulatory cells [Maccario, 2005 #18; Beyth, 2005 #38; Aggarwal, 2005 #43] and inhibitory effects on maturation of dendritic cells [Zhang, 2004 #44; Aggarwal, 2005 #43; Maccario, 2005 #18; Jiang, 2005 #45; Nauta, 2006 #42].
Whether or not MSC can induce tolerance in allogeneic transplantation setting is still an area of debate [Zappia, 2005 #108; Barry, 2005 #88; Nauta, 2006 #42], however, decreased GvHD in allogenic stem cell transplantation and treatment of acute GvHD in vivo have been demonstrated [Le Blanc, 2004 #1; Le Blanc, 2006 #50].
We and others have shown that cells isolated from the mesenchymal region of human amnion and chorion fail to induce allogenic T cell responses and actively suppress T cell proliferation induced by alloantigens [Bailo, 2004 #31; Wolbank, 2007 #86]. Furthermore, we have shown that a heterogeneous population isolated from the human amnion and chorion fetal membranes demonstrated long term chimerism in xenogenic animal transplantation models, suggesting their reduced immunogenicity and tolerogenic potential [Bailo, 2004 #31].
It is tempting to speculate that the immunomodulatory characteristics of the mesenchymal cells resident within the fetal membranes play a role in the fetal-maternal tolerance process, however this theory remains to be proven.
Scientists have long been puzzled by the mechanisms involved in maternal tolerance to the fetus. Proposed explanations are the anatomical barrier between the mother and the fetus formed by the placenta, the immunologic inertness of the mother and the antigenic immaturity of the fetus [Medawar, 1953 #103]. However, several studies have indicated that the fetal placental barrier may be less inert or impervious than previously envisioned and presented evidence for cellular trafficking in both directions across the fetal/maternal interface [Collins, 1980 #102, Bonney, 1997 #80, Bianchi, 2003 #99]. In addition, it is now clear that the maternal immune system is not anergic to all fetal tissues since it can respond and eliminate fetal cells that enter the maternal circulation [Bonney, 1997 #80; Bonney, 1998 #82]. Finally, it is well accepted that fetal trophoblast cells lack the major histocompatibility complex (MHC) class II (MHC-II) antigens, downregulate MHC class I proteins, and express high levels of HLA-G, an antigen known to protect against rejection [Koch, 2003 #105]. However, fundamental questions that still remain are whether the fetus participates actively in suppressing maternal allogeneic immune responses and, if so, what fetal placental tissues play an immunomodulatory role. The mesodermal (stromal) layers of amnion and chorion are considered avascular and therefore inert in terms of immune presentation, however, macrophage-like populations in the chorion (Hofbauer cells) have been described in previous reports [Enders, 1970 #113]. More recently a defined population of HLA-DR-expressing cells with macrophage-monocyte phenotypic characteristics has also been described in the mesenchymal layer of the amnion [Sutton, 1986 #83; Sutton, 1983 #84; Bulmer, 1988 #85] thus suggesting the presence of populations capable of active immune function within these tissues.
In this study we set out extend our investigations on the immunomodulatory characteristics of amniotic cells and discovered that subfractions of cells isolated from the amniotic mesenchymal tissue can indeed induce either inhibitory or stimulating effects on allogeneic T lymphocytes.
It was the object of the present invention to provide a method for isolating and culturing cells that have an effect on the immune system. Moreover, it was the object of the present invention to provide medicaments that can be used to modulate, stimulate, suppress or otherwise influence the immune response. Moreover, it was the object to provide a medicament to avoid transplant rejection.
These objects are solved by the methods disclosed and the cells obtainable therewith as well as the medicaments and their use.
The inventors found that useful cells can be isolated from the amnion, chorion and umbilical cord.